Viral neutralizing antibody (NtAb) epitope mapping can assist in the development of new vaccines and pharmaceuticals for the prevention and/or treatment of infectious diseases. Additionally, viral NtAb epitope mapping can assist in the development of gene delivery vectors. Identification of and knowledge regarding viral NtAb epitopes may help in the genetic engineering of components of viral vectors that may evade a host immune response, as the host immune response can be a significant obstacle to effective in vivo gene therapy.
Adeno-associated virus (AAV) is a promising in vivo gene delivery vector for gene therapy. Various issues remain to be overcome, however, in the use of AAV as an in vivo gene delivery vector, including the requirement of high vector dose for clinically beneficial outcomes, efficacy-limiting host immune response against viral proteins, promiscuous viral tropism, and the high prevalence of pre-existing anti-AAV neutralizing antibodies in humans. Despite these issues, interest in the use of AAV in gene therapy is growing. A number of naturally occurring serotypes and subtypes have been isolated from human and non-human primate tissues (Gao G et al., J Virol 78, 6381-6388 (2004) and Gao G et al., Proc Natl Acad Sci USA 99, 11854-11859 (2002), both of which are incorporated by reference herein). Among the newly-identified adeno-associated virus isolates, AAV serotype 8 (AAV8) and AAV serotype 9 (AAV9) have gained much attention because recombinant adeno-associated vectors (rAAVs) derived from these two serotypes can transduce various organs including the liver, heart, skeletal muscles, and central nervous system with high efficiency following systemic administration via the periphery (Foust K D et al., Nat Biotechnol 27, 59-65 (2009); Gao et al., 2004, supra; Ghosh A et al., Mol Ther 15, 750-755 (2007); Inagaki K et al., Mol Ther 14, 45-53 (2006); Nakai H et al., J Virol 79, 214-224 (2005); Pacak C A et al., Circ Res 99, e3-e9 (2006); Wang Z et al., Nat Biotechnol 23, 321-328 (2005); and Zhu T et al., Circulation 112, 2650-2659 (2005), all of which are incorporated by reference herein).
The robust transduction by rAAV8 and rAAV9 vectors has been presumed to be ascribed to strong tropism for these cell types, efficient cellular uptake of vectors, and/or rapid uncoating of virion shells in cells (Thomas C E et al., J Virol 78, 3110-3122 (2004), incorporated by reference herein). In addition, emergence of capsid-engineered rAAV with better performance has significantly broadened the utility of rAAV as a vector toolkit (Asokan A et al., Mol Ther 20, 699-708 (2012), incorporated by reference herein). Proof-of-concept for rAAV-mediated gene therapy has been shown in many preclinical animal models of human diseases. Phase I/II clinical studies have been initiated or completed for genetic diseases including hemophilia B (Manno C S et al., Nat Med 12, 342-347 (2006) and Nathwani A C et al., N Engl J Med 365, 2357-2365 (2011), both of which are incorporated by reference herein); muscular dystrophy (Mendell J R et al., N Engl J Med 363, 1429-1437 (2011), incorporated by reference herein); cardiac failure (Jessup M et al., Circulation 124, 304-313 (2011), incorporated by reference herein); blinding retinopathy (Maguire A M et al., Lancet 374, 1597-1605 (2009), incorporated by reference herein); and al anti-trypsin deficiency (Flotte T R et al., Hum Gene Ther 22, 1239-1247 (2011), incorporated by reference herein), among others.
Although rAAV vectors have widely been used in preclinical animal studies and have been tested in clinical safety studies, the current rAAV-mediated gene delivery systems remain suboptimal for broader clinical applications. The sequence of an AAV viral capsid protein defines numerous features of a particular AAV vector. For example, the capsid protein affects features such as capsid structure and assembly, interactions with AAV nonstructural proteins such as Rep and AAP proteins, interactions with host body fluids and extracellular matrix, clearance of the virus from the blood, vascular permeability, antigenicity, reactivity to NtAbs, tissue/organ/cell type tropism, efficiency of cell attachment and internalization, intracellular trafficking routes, and virion uncoating rates. Furthermore, the relationship between a given AAV capsid amino acid sequence and the characteristics of the rAAV vector are unpredictable.
High prevalence of pre-existing NtAbs against AAV capsids in humans poses a significant barrier to successful AAV vector-mediated gene therapy. There has been strong enthusiasm about developing “stealth” AAV vectors that can evade NtAbs; however, creation of such AAVs requires more comprehensive information about NtAb epitopes, which currently remains very limited.
DNA-barcoded AAV2R585E hexapeptide (HP) scanning capsid mutant libraries have been produced in which AAV2-derived HPs were replaced with those derived from other serotypes. These libraries have been injected intravenously into mice harboring anti-AAV1 or AAV9 capsid antibodies, which has led to the identification of 452-QSGSAQ-457 (SEQ ID NO:1) in the AAV1 capsid and 453-GSGQN-457 (SEQ ID NO:2) in the AAV9 capsid as epitopes for anti-AAV NtAbs in mouse sera (Adachi K et al., Nat Commun 5, 3075 (2014)). These epitopes correspond to the highest peak of the three-fold symmetry axis protrusion on the capsid. In addition, this region may also function as an epitope for mouse anti-AAV7 NtAbs using the same in vivo approach. A sequencing-based high-throughput approach, termed AAV Barcode-Seq, can allow characterization of phenotypes of hundreds of different AAV strains and can be applied to anti-AAV NtAb epitope mapping.